Improved Sézary cell detection and novel insights into immunophenotypic and molecular heterogeneity in Sézary syndrome

Sézary syndrome (SS) is an aggressive leukemic form of cutaneous T-cell lymphoma with neoplastic CD4+ T cells present in skin, lymph nodes, and blood. Despite advances in therapy, prognosis remains poor, with a 5-year overall survival of 30%. The immunophenotype of Sézary cells is diverse, which hampers efficient diagnosis, sensitive disease monitoring, and accurate assessment of treatment response. Comprehensive immunophenotypic profiling of Sézary cells with an in-depth analysis of maturation and functional subsets has not been performed thus far. We immunophenotypically profiled 24 patients with SS using standardized and sensitive EuroFlow-based multiparameter flow cytometry. We accurately identified and quantified Sézary cells in blood and performed an in-depth assessment of their phenotypic characteristics in comparison with their normal counterparts in the blood CD4+ T-cell compartment. We observed inter- and intrapatient heterogeneity and phenotypic changes over time. Sézary cells exhibited phenotypes corresponding with classical and nonclassical T helper subsets with different maturation phenotypes. We combined multiparameter flow cytometry analyses with fluorescence-activated cell sorting and performed RNA sequencing studies on purified subsets of malignant Sézary cells and normal CD4+ T cells of the same patients. We confirmed pure monoclonality in Sézary subsets, compared transcriptomes of phenotypically distinct Sézary subsets, and identified novel downregulated genes, most remarkably THEMIS and LAIR1, which discriminate Sézary cells from normal residual CD4+ T cells. Together, these findings further unravel the heterogeneity of Sézary cell subpopulations within and between patients. These new data will support improved blood staging and more accurate disease monitoring

Exploring the IL-21–STAT3 Axis as Therapeutic Target for Sézary Syndrome

Sézary syndrome is an aggressive cutaneous T-cell lymphoma. The malignant cells (Sézary cells) are present in skin, lymph nodes, and blood, and express constitutively activated signal transducer and activator of transcription (STAT)3. STAT3 can be activated by IL-21 in vitro and the IL-21 gene itself is a STAT3 target gene, thereby creating an autocrine positive feedback loop that might serve as a therapeutic target. Sézary cells underwent apoptosis when incubated with Stattic, a selective STAT3 inhibitor. STAT3 activation in Sézary cells did not affect expression of the supposed anti-apoptotic STAT3 target genes BCL2, BCL-xL, and SURVIVIN, whereas expression of (proto)oncogenes miR-21, TWIST1, MYC, and PIM1 was significantly increased. CD3/CD28-mediated activation of Sézary cells induced IL-21 expression, accompanied by STAT3 activation and increased proliferation. Blocking IL-21 in CD3/CD28-activated cells had no effects, whereas Stattic abrogated IL-21 expression and cell proliferation. Thus, specific inhibition of STAT3 is highly efficient in the induction of apoptosis of Sézary cells, likely mediated via the regulation of (proto)oncogenes. In contrast, blocking IL-21 alone seems insufficient to affect STAT3 activation, cell proliferation, or apoptosis. These data provide further insights into the pathogenic role of STAT3 in Sézary syndrome and strengthen the notion that STAT3 represents a promising therapeutic target in this disease

Prognostic Significance of Promoter Hypermethylation and Diminished Gene Expression of SYNPO2 in Melanoma.

status: publishe

A lack of Birbeck granules in Langerhans cells is associated with a naturally occurring point mutation in the human Langerin gene.

Item does not contain fulltextA heterozygous mutation in the Langerin gene corresponding to position 837 in the Langerin mRNA was identified in a person deficient in Birbeck granules (BG). This mutation results in an amino acid replacement of tryptophan by arginine at position 264 in the carbohydrate recognition domain of the Langerine protein. Expression of mutated Langerin in human fibroblasts induces tubular-like structures that are negative for BG-specific antibodies and do not resemble the characteristic structural features of BG

Epigenomic Analysis of Sézary Syndrome Defines Patterns of Aberrant DNA Methylation and Identifies Diagnostic Markers

Sézary syndrome (Sz) is a malignancy of skin-homing CD4+ memory T cells that is clinically characterized by erythroderma, lymphadenopathy, and blood involvement. Distinction of Sz from erythroderma secondary to inflammatory skin diseases (erythrodermic inflammatory dermatosis [EID]) is often challenging. Recent studies identified recurrent mutations in epigenetic enzymes involved in DNA modification in Sz. Here we defined the DNA methylomes of purified CD4+ T cells from patients with Sz, EID, and healthy control subjects. Sz showed extensive global DNA methylation alterations, with 7.8% of 473,921 interrogated autosomal CpG sites showing hypomethylation and 3.2% hypermethylation. Promoter CpG islands were markedly enriched for hypermethylation. The 126 genes with recurrent promoter hypermethylation in Sz included multiple candidate tumor suppressors that showed transcriptional repression, implicating aberrant methylation in the pathogenesis of Sz. Validation in an independent sample set showed promoter hypermethylation of CMTM2, C2orf40, G0S2, HSPB6, PROM1, and PAM in 94–100% of Sz samples but not in EID samples. Notably, promoter hypermethylation of a single gene, the chemokine-like factor CMTM2, was sufficient to accurately distinguish Sz from EID in all cases. This study shows that Sz is characterized by widespread yet distinct DNA methylation alterations, which can be used clinically as epigenetic diagnostic markers

A Meta-Analysis of Gene Expression Data Identifies a Molecular Signature Characteristic for Tumor-Stage Mycosis Fungoides

Mycosis fungoides (MF) is the most common type of primary cutaneous T-cell lymphoma (CTCL). To identify a molecular signature characteristic of MF tumor stage, we used a bioinformatic approach involving meta-analysis of publicly available gene expression data sets combined with previously generated gene expression data. Results for a selection of genes were further refined and validated by quantitative PCR and inclusion of additional controls. With this approach, we identified a profile specific for MF tumor stage, consisting of 989 aberrantly expressed genes, the majority of which (718 genes) are statistically significantly more expressed in MF compared with normal skin, inflamed skin, and normal T cells. As expected, the signature contains genes reflecting the highly proliferative characteristic of this T-cell malignancy, including altered expression of cell cycle and kinetochore regulators. We uncovered details of the immunophenotype, suggesting that MF originates from IL-32-producing cells and identified previously unreported therapeutic targets and/or diagnostic markers, for example, GTSF1 and TRIP13. Loss of expression of the NF-κB inhibitor, NFKBIZ, may partly explain the enhanced activity of NF-κB, which is a hallmark of MF and other CTCLs